IndiSpin QIAcube HT Pathogen Kit Handbook...CAUTION: DO NOT add bleach or acidic solutions directly to the sample preparation waste. Bildergebnis für handbook icon IndiSpin QIAcube

Jan 2021 | EN

IndiSpin® QIAcube® HT Pathogen Kit Handbook

For automated purification of viral RNA and DNA and bacterial DNA from animal whole blood, serum, plasma, body fluids, oral fluids, swabs and washes, tissue and feces

IndiSpin QIAcube HT Pathogen Kit (cat no SP54161),

formerly cador® Pathogen 96 QIAcube HT Kit

Manufactured by QIAGEN® GmbH for INDICAL BIOSCIENCE

INDICAL BIOSCIENCE GmbH, Deutscher Platz 5b,

04103 Leipzig, Germany

2 IndiSpin QIAcube HT Pathogen Kit Handbook 01/2021

Contents

Kit contents ............................................................................................. 4

Storage ................................................................................................... 5

Intended use ........................................................................................... 5

Symbols .................................................................................................. 6

Safety information ................................................................................... 6

Quality control ......................................................................................... 7

Introduction ............................................................................................. 8

Principle and procedure .......................................................................... 8

Nucleic acid purification protocol ............................................................ 9

Pretreatments ....................................................................................... 11

Equipment and reagents to be supplied by user .................................. 13

Important notes ..................................................................................... 14

Starting material ................................................................................ 14

Yields of nucleic acids ...................................................................... 17

Using Carrier RNA and internal controls .......................................... 17

Storing nucleic acids ......................................................................... 18

Handling RNA ................................................................................... 19

Preparing reagents ........................................................................... 19

Assembling the vacuum chamber .................................................... 23

Optional features .................................................................................. 25

Processing fewer than 96 samples per run ...................................... 25

Off-board lysis ................................................................................... 25

Manual lysis procedure ..................................................................... 26

Protocol for use with QIAcube HT 4.17 software.................................. 27

Important points before starting ........................................................ 27

IndiSpin QIAcube HT Pathogen Kit Handbook 01/2021 3

Things to do before starting .............................................................. 27

Procedure ......................................................................................... 29

Cleaning the instrument after completing a run ................................ 30

Protocol for use with QIAcube HT Prep Manager software .................. 31

Important points before starting ........................................................ 31

Things to do before starting .............................................................. 31

Procedure ......................................................................................... 33

Cleaning the instrument after completing a run ................................ 34

Optional steps ................................................................................... 35

Troubleshooting guide .......................................................................... 40

Order information .................................................................................. 43

Change index ........................................................................................ 44


Kit contents

IndiSpin QIAcube HT Pathogen Kit

Cat. no.

Number of preps

(480)

SP54161

480

IndiSpin 96 Plate 5

Buffer VXL1 3 x 30 ml

Buffer ACB (concentrate)1,2 2 x 60 ml

Proteinase K 2 x 6 ml

Carrier RNA (poly A) 2 x 310 µg

Buffer AW1 (concentrate)1,3 2 x 98 ml

Buffer AW2 (concentrate)3 2 x 66 ml

Buffer AVE 2 x 125 ml

TopElute Fluid 1 x 60 ml

Quick-Start Protocol (PCard) 2

1 CAUTION: Contains a chaotropic salt. Take appropriate laboratory safety measures and wear gloves when handling. Not compatible with disinfectants containing bleach. See page 5 for safety information. 2 Before using for the first time, add isopropanol as indicated on the bottle to obtain a working solution. 3 Before using for the first time, add ethanol (96-100%) as indicated on the bottle to obtain a working solution.


Storage

IndiSpin 96 plates and buffers can be stored dry at room temperature

(15-25ºC) until the expiration date stated on the kit box without affecting

performance.

Lyophilized Carrier RNA can be stored at room temperature until the

expiration date stated on the kit box. For use, lyophilized Carrier RNA

should be dissolved in Buffer AVE and then added to Buffer VXL, as

described in “Preparing reagents”, on page 19. This Carrier RNA/Buffer

AVE/Buffer VXL mix solution should be prepared fresh and is stable at

room temperature for up to 48 hours. Unused Carrier RNA dissolved in

Buffer AVE should be immediately frozen in aliquots at -30 to -15°C. Do

not subject aliquots of Carrier RNA to more than 3 freeze-thaw cycles.

Proteinase K can be stored at room temperature (15-25°C). To store for

extended periods of time, or if the ambient temperature often exceeds

25°C, we recommend storing at 2-8°C.

Intended use

The IndiSpin QIAcube HT Pathogen Kit is intended for the extraction of

pathogen nucleic acids (viral RNA and DNA, and bacterial DNA) from

animal whole blood, serum, plasma, body fluids, oral fluids, swabs,

washes, tissue (homogenate), and feces.

For molecular biology applications.


Symbols

Legal manufacturer

Lot number

Use by date

Temperature limitations for storage

Handbook

Catalog number

Material number

Safety information

When working with chemicals, always wear a suitable lab coat,

disposable gloves and protective goggles. For more information, please

consult the appropriate safety data sheets (SDSs). These are available

from your local sales representative or by Email request under

[email protected].

CAUTION: DO NOT add bleach or acidic solutions

directly to the sample preparation waste.

mailto:[email protected]

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Buffer VXL and Buffer AW1 contain guanidine hydrochloride, and Buffer

ACB contains guanidine thiocyanate, which can form highly reactive

compounds if combined with bleach.

If liquid containing these buffers is spilled, clean with suitable laboratory

detergent and water. If the spilled liquid contains potentially infectious

agents, clean the affected area first with laboratory detergent and

water, and then with 1% (v/v) sodium hypochlorite.

Quality control

In accordance with INDICAL’s ISO-certified Quality Management

System, each lot of IndiSpin QIAcube HT Pathogen Kit is tested against

predetermined specifications to ensure consistent product quality.


Introduction

The IndiSpin QIAcube HT Pathogen Kit enables the efficient purification

of viral RNA and DNA, as well as bacterial DNA, from a broad range of

animal sample types, including whole blood, serum, plasma, swabs,

washes, tissue, and feces (see “Starting material” on page 14). The

extracted nucleic acids are free of proteins, nucleases, and other

impurities, and are ready for use in downstream applications, such as

real-time PCR-based pathogen identification.

However, specific combinations of sample types and pathogens should

be validated by the user.

The kit is not intended for host RNA or host DNA preparation.

Principle and procedure

Samples are lysed under highly denaturing conditions at room

temperature (15-25°C) in the presence of Proteinase K and Buffer VXL,

which together ensure the inactivation of nucleases. Adding Buffer ACB

adjusts the binding conditions for the copurification of DNA and RNA.

The lysate is then transferred to an IndiSpin 96 Plate and coated with

ethanol. During vacuum, nucleic acids are adsorbed onto the silica

membranes while contaminants pass through. Three efficient wash

steps remove the remaining contaminants and enzyme inhibitors, and

nucleic acids are eluted in Buffer AVE.

Performance is not guaranteed for every combination of starting

material and pathogen species and must be validated by the user.

Some samples may require a pretreatment (see Table 1, page 10).


Nucleic acid purification protocol

This handbook contains two protocols describing the purification of

pathogen nucleic acids from different sample types:

• For use with QIAcube HT 4.17 software (starting page 27).

• For use with QIAcube HT Prep Manager (starting page 31).

The purification of pathogen nucleic acids is optimized for purification of

viral RNA and DNA, and the DNA of easy-to-lyse bacteria from up to

200 μl of fluid material. Suitable starting materials for direct processing

using this method include:

• whole blood

• serum

• plasma

• oral fluid

• body cavity fluids (e.g., peritoneal, synovial, cerebrospinal)

• liquid extracts from swabs (e.g., nasal, pharyngeal, and

cloacal* swabs)

• wash fluids (e.g., from bronchoalveolar lavages)

• other fluids, such as urine or feces suspensions*

Most sample types can be directly processed without pretreatment.

However, depending on the starting material and the target pathogen,

one of the pretreatment protocols may be needed. For samples that

require a pretreatment prior to nucleic acid purification, Table 1 on

page 11 provides an overview of which pretreatment protocols are

suited to different starting material and pathogen combinations.

* The processing of samples with a high inhibitor content, such as urine and feces, may require a reduction in sample input volume or further measurements. For further pretreatment recommendations, contact INDICAL support ([email protected]).


The lysis and binding solutions used in the procedure are Buffer VXL

and Buffer ACB. Please pay attention to the information given under

“Safety information”, page 6.


Pretreatments

The pretreatments mentioned in this handbook are optimized for

specific combinations of starting material and target pathogens.

The choice of pretreatment depends on the workflow focus and is to be

followed by nucleic acid purification.

Table 1 on page 11 summarizes the pretreatments and their

applications.

Some of the pretreatments may require additional components, which

are indicated in each pretreatment protocol.

Table 1: Overview of available pretreatment protocols

Sample Target Pretreatment handbook

Fluids

(e.g., whole blood,

serum, plasma,

swab or wash

fluid, pretreated

tissue)

Viral RNA and

DNA, DNA of

easy-to-lyse

bacteria1

- -

Whole blood or

pretreated tissue

DNA of difficult-

to-lyse bacteria1

Pretreatment B1

for difficult-to-lyse bacteria in

whole blood or pretreated

tissue

HB-2533

Serum, plasma,

swabs, washes,

body cavity fluids,

urine

DNA of difficult-

to-lyse bacteria1

Pretreatment B2


body fluids2

HB-2534

High volume cell-

free fluids (for

increased

sensitivity)

DNA of easy-to-

lyse bacteria1

Pretreatment B3


body fluids2

HB-2549


Tissue

(e.g., liver, spleen,

kidney, lymph

node)

Pathogen

nucleic acids

Pretreatment T1

Mechanical disruption of

tissue

HB-2535

Viral DNA3,

bacterial DNA4

Pretreatment T2

Enzymatic digestion of tissue

HB-2536

Rapid Partial

Disruption of

tissue

Viral RNA and

DNA,

DNA of easy-to-

lyse bacteria1

Pretreatment T3

HB-2537

Tissue containing

high amount of

lipids and/or

nucleases (e.g.

brain, pancreas)

Viral RNA and

DNA,

DNA of easy-to-

lyse bacteria1

Pretreatment T4

HB-2538

Feces Viral RNA and

DNA

Pretreatment F1

Non-lysing suspension

method

HB-2513

Bacterial DNA1

and viral DNA

Pretreatment F2

Lysing suspension method

HB-2514

Mycobacterium

avium subsp.

paratuberculosis

(MAP) DNA

Pretreatment F-MAP HB-2503

Filter paper cards Pretreatment C1 HB-2520

Swabs (tracheal,

oropharyngeal,

blood)

Pretreatment S1 HB-2516

Oral fluids Pretreatment O1 HB-2551

1 Gram-positive bacteria are difficult to lyse due to their rigid cell wall. Many Gram-negative bacteria are easy to lyse, but some are not and will also benefit from Pretreatment B1 or B2. 2 Not suitable for whole blood. 3 Not suitable for viral RNA as the lysis conditions do not sufficiently conserve RNA integrity. 4 For difficult-to-lyse bacteria, subsequently use Pretreatment B1.

For further information on Pretreatments website

www.indical.com/handbooks contact INDICAL Support at

[email protected].


Equipment and reagents to be supplied by user

When working with chemicals, always wear a suitable lab coat,

disposable gloves and protective goggles. For more information,

consult the appropriate safety data sheets (SDSs), available from the

product supplier.

• Pipettors and disposable pipette tips with aerosol barriers

(20-1000 μl)

• Ethanol (96-100%)*

• Isopropanol

• Phosphate-buffered saline (PBS), may be required for diluting

samples

• Vortexer

• QIAcube HT instrument

• QIAcube HT Plasticware

• QIAcube HT 4.17 Software or QIAcube HT Prep Manager Software

• QIAcube HT Reagent troughs

* Do not use denatured alcohol, which contains other substances such as methanol or

methylethylketone.


Important notes

Starting material

Do not overload the IndiSpin 96 Plate, as this can lead to impaired

nucleic acid extraction and/or performance in downstream assays. For

samples with very high host nucleic acid contents (e.g., for certain

tissues, such as spleen or blood samples with highly increased cell

counts), use less than the maximum amount of sample recommended

in the protocol or pretreatments. In some downstream applications such

as PCR and RT-PCR, very high background concentrations of nucleic

acids may impair the reaction. Use appropriate controls (e.g., an

internal control) to verify successful PCR amplification.

Avoid transferring solid material to the S-Block as this can reduce flow

through the membrane (e.g., blood clots, solid tissue, swab fibers).

When working with difficult samples, use a vacuum performance check

to check if all liquid has passed the membrane.

Highly viscous fluids may require treatment to reduce their viscosity, to

allow for efficient extraction of pathogen nucleic acids. Please contact

INDICAL support at [email protected] for recommendations.

Avoid repeated thawing and freezing of samples, since this may reduce

nucleic acid yield and quality.

Animal whole blood

Blood samples treated with EDTA, citrate, or heparin as anticoagulant

can be used for nucleic acid purification. Samples can be either fresh or

frozen, provided that they have not been freeze-thawed more than

once. Freeze-thawing more than once can lead to denaturation and


precipitation of proteins, resulting in potential reduction in viral titers,

and therefore, reduced yields of viral nucleic acids.

After collection and centrifugation, whole blood samples can be stored

at 2-8ºC for up to 6 hours. For longer storage, we recommend freezing

aliquots at -30 to -15ºC or at -70ºC.

We recommend using 50-200 μl blood containing non-nucleated

erythrocytes. However, highly elevated cell counts due to inflammatory

or neoplastic diseases may strongly increase the host nucleic acid

content of a sample. In this case, reduction of sample input to 50 μl

may improve results in downstream assays, particularly in RT-PCR.

If using less than 200 μl blood, adjust the sample volume to 200 μl with

PBS or 0.9% NaCl.

For blood samples containing nucleated erythrocytes (e.g., samples

from bird and fish), use less than 50 μl blood and adjust the sample

volume to 200 μl with PBS or 0.9% NaCl.

Animal serum, plasma, other body fluids, swabs, oral fluids, and wash

specimens

Frozen plasma or serum must not be thawed more than once before

processing.

We recommend storing swabs in transport media; for example, viral

transport media (VTM) or brain-heart infusion broth (BHI). Remove the

swab and squeeze out the liquid by pressing the swab against the

inside of the storage tube. For extraction of viral RNA or DNA, we

recommend centrifuging the swab media briefly to ensure any residual

solid materials are removed.

Note: Solid pieces remaining in the sample fluid may aggregate on the

IndiSpin 96 Plate membrane, which may decrease nucleic acid yield.


Up to 200 μl serum, plasma, other body fluid, swab media supernatant,

or wash fluid can be processed.

Carrier RNA must be used in the nucleic acid purification protocol to

prevent the loss of nucleic acids during the procedure (see page 17 for

information on the use of Carrier RNA).

The processing of samples with very high inhibitor contents, such as

urine or fecal suspensions, may require a reduction in sample input

volume and/ or an extra pretreatment to remove inhibitors. To reduce

the input volume, use 25-50 μl of the sample and adjust the volume to

200 μl with PBS or 0.9% NaCl.

For extraction of bacterial DNA, the input volume can be increased to

more than 200 μl, e.g., 1.5 ml for increased sensitivity of bacterial

detection. Gram-negative bacteria in cell-free fluids can be

concentrated by centrifugation of higher volumes. Resuspend pellets in

PBS and use 200 μl as starting volume. See Pretreatment B2 for

extraction of DNA from difficult-to-lyse bacteria.

Animal tissues

When working with tissue samples, mechanical or enzymatic disruption

of the tissue structure is the prerequisite for liberation of cells,

subsequent release of nucleic acids, and membrane permeability of the

material.

Different tissue types can vary widely with regard to texture and rigidity,

cell types, and content of host nucleic acids and inhibitory substances.

In addition, the localization of pathogen nucleic acids in the tissue may

vary depending on tissue type, pathogen, and stage of infection.

Therefore, suitability of the pretreatment protocols in this handbook

should be evaluated for each new combination of tissue and pathogen.

Additional pretreatments for tissue samples are available at INDICAL

Support, including a rapid protocol and recommendations for difficult

tissues.


Up to 25 mg of fresh or frozen tissue can be used as a starting amount.

For tissues with a very high number of cells for a given mass of tissue,

such as spleen, a reduced amount of starting material (5-10 mg) should

be used.

Using too much input material decreases the quality and the amount of

DNA and leads to an increased risk of blocked membranes.

Yields of nucleic acids

For samples containing a low amount of cells (e.g., serum and plasma),

the yield of viral nucleic acids obtained can be below 1 μg and is

therefore difficult to quantify using a spectrophotometer. In addition,

eluates prepared with Carrier RNA may contain much more Carrier

RNA than target nucleic acids. The IndiSpin QIAcube HT Pathogen Kit

recovers total nucleic acids. Therefore, cellular DNA and RNA will be

co-purified from any cells in the sample along with viral RNA and DNA,

and bacterial DNA, and cannot be distinguished using

spectrophotometric measurements. We recommend using quantitative

amplification methods such as quantitative real-time PCR or real-time

RT-PCR to determine pathogen nucleic acid yields.

Using Carrier RNA and internal controls

Carrier RNA

We recommend adding Carrier RNA to fluids containing low amounts of

cells such as serum, plasma, swab media, and wash fluid.

This enhances adsorption of viral RNA and DNA to the silica

membranes, which is especially important when the target molecules

are not abundant. In addition, an excess of Carrier RNA reduces the

chances of viral RNA degradation in the rare event that RNases are not

denatured by the chaotropic salts and detergents in the lysis buffer. Not


using Carrier RNA may decrease the recovery of viral nucleic acids. Do

not add Carrier RNA to whole blood and tissue samples, or other

samples containing a high amount of cells.

Internal Control

Use of an internal control, such as the intype IC-DNA or intype IC-RNA

is optional, depending on the amplification system of choice. If the

IndiSpin QIAcube HT Pathogen Kit is used in combination with

amplification systems that employ an internal control, introduction of

these internal controls may be required during the purification

procedure, to monitor the efficiency of sample preparation and

downstream assay.

Add unprotected internal control nucleic acids (e.g., plasmid DNA or in

vitro transcribed RNA) to VXL mixture only. Do not add these internal

control nucleic acids directly to the sample.

The amount of internal control added depends on the assay system

and the elution volume. Evaluation of the correct amount of internal

control nucleic acid must be performed by the user. Refer to the

manufacturer’s instructions to determine the optimal concentration of

internal control or contact INDICAL Support ([email protected]) for

further information.

Storing nucleic acids

For short-term storage of up to 24 hours, we recommend storing the

purified viral RNA and DNA at 2-8°C. For storage longer than 24 hours,

we recommend storing purified nucleic acids at -30 to -15°C, or even at

-70°C in the case of RNA.


Handling RNA

RNases are very stable and active enzymes that generally do not

require cofactors to function. Since RNases are difficult to inactivate

and only minute amounts are sufficient to destroy RNA, do not use any

plasticware or glassware without first eliminating possible RNase

contamination. Care should be taken to avoid inadvertently introducing

RNases into the RNA sample during or after the purification procedure.

Preparing reagents

Carrier RNA stock solution

For use, lyophilized Carrier RNA should first be dissolved in Buffer

AVE. Add 310 μl Buffer AVE to the tube containing 310 μg lyophilized

Carrier RNA to obtain a stock solution of 1 μg/μl. Add this solution to

Buffer VXL mixture as in Table 2 on page 28 or Table 3 page 32.

Unused Carrier RNA dissolved in Buffer AVE should be frozen in

aliquots at -30 to -15°C. Aliquots of Carrier RNA should not be

subjected to more than 3 freeze-thaw cycles.

Adding Carrier RNA to Buffer VXL

We recommend adding Carrier RNA to fluids containing a low amount

of cells such as serum, plasma, swabs media, and wash fluid. Do not

add Carrier RNA to samples with a high cell content such as whole

blood and tissue because high amounts of background nucleic acids

may negatively influence downstream applications such as RT-PCR.

Carrier RNA dissolved in Buffer AVE is added to Buffer VXL so that

1 μg carrier RNA is present in each sample.


Note: 100 μl of Buffer VXL containing dissolved Carrier RNA is used

per preparation.

Important note: Carrier RNA does not dissolve in Buffer VXL. It must

first be dissolved in Buffer AVE.

The Buffer VXL solution containing dissolved Carrier RNA should be

prepared fresh and is stable at room temperature (15-25ºC) for up to 48

hours.

Proteinase K

The IndiSpin QIAcube HT Pathogen Kit contains ready-to-use

Proteinase K supplied in a specially formulated storage buffer. The

activity of the Proteinase K solution is 600 mAU/ml.

Proteinase K is stable for at least 1 year after delivery when stored at

room temperature (15-25°C). To store for more than 1 year or if

ambient temperature often exceeds 25°C, we recommend storing

Proteinase K at 2-8°C.

Add Proteinase K to Buffer VXL immediately before starting the

protocol.

Buffer ACB

Buffer ACB is supplied as a concentrate. Before using for the first time,

add Isopropanol (100%) as indicated on the bottle. Tick the check box

on the bottle label to indicate that Isopropanol has been added. Mix well

after adding Isopropanol.


Buffer AW1

Buffer AW1 is supplied as a concentrate. Before using for the first time,

add Ethanol (96-100%) as indicated on the bottle. Tick the check box

on the bottle label to indicate that ethanol has been added.

Reconstituted Buffer AW1 can be stored at room temperature

(15-25°C) for up to 1 year. Mix well after adding Ethanol.

Buffer AW2

Buffer AW2 is supplied as a concentrate. Before using for the first time

add Ethanol (96-100%) as indicated on the bottle. Tick the check box

on the bottle label to indicate that ethanol has been added. Mix well

after adding Ethanol.

Handling Buffer AVE

Buffer AVE is RNase-free upon delivery. It contains sodium azide, an

antimicrobial agent that prevents growth of RNase-producing

organisms. However, as this buffer does not contain any RNase-

degrading chemicals, it will not actively inhibit RNases introduced by

inappropriate handling. When handling Buffer AVE, take extreme care

to avoid contamination with RNases. Follow general precautions for

working with RNA, such as frequent change of gloves and keeping

tubes closed whenever possible.

TopElute Fluid

TopElute Fluid is used during elution of nucleic acids from the IndiSpin

96 Plate membrane. It enables application of a stable and high vacuum

and results in equal eluate volumes. In addition, TopElute Fluid

eliminates the formation of drops of elution buffer at the outlet nozzles

of the IndiSpin 96 Plate.


TopElute Fluid might be found as a top layer over the elution buffer. It is

inert and has no effects on downstream applications.

Important: Please ensure that you only take the eluate from below the

top layer.


Assembling the vacuum chamber

Figure 1 on page 24 illustrates the assembly of the vacuum chamber.

For further information, please refer to the QIAcube HT User Manual.

1. Insert the channeling block holder into the left (waste) chamber of

the vacuum chamber.

2. Press firmly on the sides of the channeling block holder to seat it in

the chamber, sealing the O-ring on the spigot into the drain.

3. Then, place the channeling block into the channeling block holder.

4. Place the IndiSpin 96 plate in the transfer carriage. Load the

carriage with the IndiSpin 96 plate into the left (waste) chamber of

the vacuum chamber.

5. Ensure that the carriage is positioned to the left inside the vacuum

chamber. Place the riser block EMTR in the right (elution) chamber

of the vacuum chamber with the pin of the riser block EMTR in the

top right position.

6. Load an elution microtubes rack (EMTR) into the elution chamber.


Figure 1: Assembling the vacuum chamber

IndiSpin 96 plate

Transfer carriage

(cat. no. 9022654,

QIAGEN)

Channeling adapter

(cat. no. 9022656,

QIAGEN)

Contains:

Channeling block

(cat. no. 9022665,

QIAGEN)

Channeling-block

holder

(cat. no. 9022664,

QIAGEN)

Elution microtubes

(EMTR)

Riser-block holder

EMTR

(cat. no. 9022655,

QIAGEN)

Vacuum chamber


Optional features

Processing fewer than 96 samples per run

If processing fewer than 96 samples reuse of IndiSpin 96 plates,

S-Block and EMTR is possible up to three times.

Note: We recommend using fresh plasticware for every run. If reusing,

take extreme care to prevent cross-contamination.

• Store plates in a way that separates the outlet nozzles under the

plate, for example, in the S-Block used in the same run or in a fresh

96-well microtiter plate.

• Cover unused wells of the S-Block and IndiSpin 96 plate with a tape

sheet at all times.

• Remove unused Elution Microtubes from the EMTR in rows of eight

tubes.

Off-board lysis

For some applications, it may be necessary to lyse samples in a safety

cabinet. For some sample types, a heated lysis outside the instrument

might enhance performance.

If lysis with Proteinase K is carried out off-board, Proteinase K may be

exchanged with water when setting up the worktable.

QIAcube HT 4.17 Software

A protocol allowing for lysis outside the instrument is available from our

technical support team.

QIAcube HT Prep Manager Software

When using an off-board lysis protocol, choose the cador pathogen


heated off-board lysis protocol in the software setup step under the

selected protocol.

Manual lysis procedure

Follow this procedure for manual sample lysis if using the cador

pathogen heated off-board lysis protocol.

1. Add 200 µl of each fluid sample to the bottom of an S-Block well.

2. Cover the S-Block with adhesive tape.

3. Incubate at 70°C, with constant agitation, for 10–15 min.

4. Optional: Briefly centrifuge the S-block to remove liquid from the

inside of the tape.

5. Remove the adhesive tape from the S-Block.

6. Proceed with the “cador pathogen protocol on the QIAcube HT”,

using either the QIAcube HT 4.17 software (page 27) or the

QIAcube HT Prep Manager software (page 31).


Protocol for use with QIAcube HT 4.17

software

This protocol is for the purification of viral RNA and DNA, and the DNA

of easy-to-lyse bacteria from fluid samples or pretreated tissue

samples.

Important points before starting

• Before beginning the procedure, read “Important notes” (page 14).

• Do not overload the IndiSpin membranes as this can lead to

impaired nucleic acid extraction and / or performance in downstream

applications.

• Avoid repeated freezing and thawing of samples as this may reduce


Things to do before starting

• If necessary, thaw and equilibrate samples at room temperature

(15-25°C).

• Check for precipitates in reagents. If a reagent contains precipitates,

incubate at 37°C with gentle shaking to dissolve precipitates. Avoid

vigorous shaking, which causes foaming.

• Check that Buffer ACB, Buffer AW1, Buffer AW2, and Carrier RNA

have been prepared according to the instructions in “Preparing

reagents” (page 19).

• When working with difficult samples, use a vacuum performance

check to check if all liquid has passed the membrane. See


Troubleshooting Guido on page 40 and the QIAcube HT User

Manual for guidance.

• Ensure that the correct software is installed.

• Ensure that the relevant version of the of the cador Pathogen 96

QIAcube HT mit EtOH 180µl VXL.QSP run file is installed on the

instrument.

• Ensure that you are familiar with operating the instrument. Refer to

the QIAcube HT User Manual for operating instructions.

• If the volume of the sample is less than 200 μl, add PBS or 0.9%

NaCl to a final volume of 200 μl.

• If necessary, prepare a mixture of Buffer VXL and Carrier RNA

according to Table 2 on page 28, for use in step 8 of the procedure.

Note: Prepare a volume of the Buffer VXL/Carrier RNA/Internal

Control mixture that is 10% greater than that required for the total

number of sample purifications to be performed.

Table 2: Buffer VXL mixture preparation

Samples 24 32 40 48 56 64 72 80 88 96

Buffer VXL (ml)

4.6 5.9 7.4 8.8 9.9 11.8 12.7 14.1 15.5 17

Proteinase K (µl)

580 740 920 1100 1240 1470 1600 1800 2000 2200

Carrier RNA* (µl)

30 40 46 56 62 74 80 90 96 100

Internal Control* (µl)

280 370 460 550 620 730 800 880 980 1000

* if you are not using the Internal Control or Carrier RNA, use RNase-free water instead.


Procedure

1. Place the tip discard chute on the worktable so that the chute is

over the tip disposal box.

2. Switch on the instrument. The switch is located at the lower left of

the backside of the instrument. Launch the QIAcube HT Software

and select the “Recent” tab.

3. To open the run file, select the protocol “cador Pathogen 96

QIAcube HT mit EtOH 180µl VXL” and then click “Open”. For more

information about optional steps and advanced options see the kit

handbook.

4. Click on the toolbar.

5. Select the appropriate number of samples arranged in columns in

the 96-well plate. Ensure that the “Turn the HEPA filter on

automatically” option is checked and click “Jump to End”. Confirm

the protocol by clicking “Finish”. The wizard closes.

6. Prepare the vacuum chamber (chapter “Assembling the vacuum

chamber”, page 23).

Note: If fewer than 12 columns are to be processed, seal unused

columns of the IndiSpin 96 plate with adhesive tape (supplied).

7. Add 200 µl sample to the selected S-Block wells. Place the S-Block

in the B1 position of the instrument’s worktable.

8. Transfer the indicated volumes of all reagents, except Buffer VXL

mixture, into the corresponding reagent troughs, close the lids, and

place them on the indicated positions on the worktable. Prepare the

indicated volume of the Buffer VXL mixture and mix well. Start the

run immediately and perform the pre-run check.

9. After completing the pre run check, close the instrument hood and

click “OK”.

10. Cover the elution plate (EMTR) with the lid and remove from the

elution chamber, when the protocol is complete.


Note: If using Top Elute Fluid, there may be 2 liquid phases in the

elution microtubes. Top Elute fluid will be the top layer over the

elution buffer.

11. Create a report (if required).

12. Discard used plasticware and the Buffer VXL mixture. We

recommend discarding leftover reagents in the reagent troughs.

13. Clean the instrument following instructions below.

Cleaning the instrument after completing a run

1. Discard racks containing only used tips.

2. Discard leftover reagents.

Note: We recommend not reusing reagents in multiple runs.

Reagents provided are sufficient for at least 10 runs of 48 samples.

Note: Do not clean the trough containing TopElute Fluid with water,

but with a dry lint-free cloth only.

3. Discard the S-Block or keep partially used blocks for reuse.

4. Remove the transfer carriage and discard the IndiSpin 96 plate or

keep partially used IndiSpin 96 plates for reuse.

5. Clean the carriage, channeling-block, channeling-block holder, and

tip chute.

6. With a damp cloth, clean any spilt reagent on the instrument

worktable or vacuum chamber.

Note: For all further cleaning and maintenance operations, see

QIAcube HT User Manual.

7. Turn on the UV lamp to decontaminate the worktable by clicking

See QIAcube HT User Manual for detailed instructions.


Protocol for use with QIAcube HT Prep

Manager software

This protocol is for the purification of viral RNA and DNA, and the DNA

of easy-to-lyse bacteria from fluid samples or pretreated tissue

samples.

Important points before starting

• Before beginning the procedure, read “Important notes” (page 14).

• Do not overload the IndiSpin membranes as this can lead to

impaired nucleic acid extraction and / or performance in downstream

applications.

• Avoid repeated freezing and thawing of samples as this may reduce


Things to do before starting

• If necessary, thaw and equilibrate samples at room temperature

(15-25°C).

• Check for precipitates in reagents. If a reagent contains precipitates,

incubate at 37°C with gentle shaking to dissolve precipitates. Avoid

vigorous shaking, which causes foaming.

• Check that Buffer ACB, Buffer AW1, Buffer AW2, and Carrier RNA

have been prepared according to the instructions in “Preparing

reagents” (page 19).

• When working with difficult samples, use a vacuum performance

check to check if all liquid has passed the membrane. See


Troubleshooting Guido on page 40 and the QIAcube HT User

Manual for guidance.

• Ensure that the correct software is installed.

• Ensure that the relevant version of the of the cador Pathogen 96 run

file is installed on the instrument.

• Ensure that you are familiar with operating the instrument. Refer to

the QIAcube HT User Manual for operating instructions.

• If the volume of the sample is less than 200 μl, add PBS or 0.9%

NaCl to a final volume of 200 μl.

• If necessary, prepare a mixture of Buffer VXL and Carrier RNA

according to Table 3 on page 32, for use in step 10 of the

procedure.

Note: Prepare a volume of the Buffer VXL/Carrier RNA/Internal

Control mixture that is 10% greater than that required for the total

number of sample purifications to be performed.

Table 3: Buffer VXL mixture preparation

Samples 24 32 40 48 56 64 72 80 88 96

Buffer VXL (ml)

4.6 5.9 7.4 8.8 9.9 11.8 12.7 14.1 15.5 17

Proteinase K (µl)

580 740 920 1100 1240 1470 1600 1800 2000 2200

Carrier RNA* (µl)

30 40 46 56 62 74 80 90 96 100

Internal Control* (µl)

280 370 460 550 620 730 800 880 980 1000

* if you are not using the Internal Control or Carrier RNA, use RNase-free water instead.


Procedure

1. Start the QIAcube HT Prep Manager software. Click on the Home

icon in the main toolbar to access the home screen.

2. Select “cador Pathogen 96” from the Create Experiment list. Follow

the instructions in the wizard and fill in all required fields.

3. In the Setup step, select Sample type and Pre-treatment for

documentation.

4. Select the protocol: “cador Pathogen protocol” (including lysis) or

“Heated off-board lysis protocol” (without lysis).

5. Define samples in the Labware selection step.

6. Arrange samples to the output plate in the Assignment step.

Note: The instrument must be switched on and connected to the

software before entering the Worktable step.

7. Follow the instructions for loading the worktable.

8. Add the volume of sample indicated in the Worktable step to the

selected S-Block wells.

9. Save the experiment by clicking the Save button in the button bar.

10. Click the Start run button to start the run.

Important: If the optional Vacuum performance check has been

selected, the software will show a dialog that needs to be confirmed

after defining vacuum steps.

11. When the protocol is complete, cover the elution plate (EMTR) with

the lid and remove it from the elution chamber.

Note: If using Top Elute Fluid, there may be 2 liquid phases in the

elution microtubes. Top Elute fluid will be the top layer over the

elution buffer.

12. Create a report (if required).

13. Follow the cleaning procedure.


Cleaning the instrument after completing a run

1. Follow the instructions in the QIAcube HT Prep Manager Software

for cleaning the instrument after a run.

2. Cover tip racks that contain only unused tips with the lid and

remove them from the worktable.

3. Cover fractions of partly used tip racks with an adhesive tape. Then

cover the tip racks with the lid and remove from the worktable.

Discard empty tip racks.

4. If the run has been stopped and the instrument did not remove all

used tips, remove them now and discard them.

5. Remove all reagent troughs and discard them.

Note: We recommend not reusing reagents for multiple runs.

6. Remove the input plate.

7. Discard the IndiSpin 96 plate or keep partially used IndiSpin 96

plates for subsequent reuse. In this case cover used fractions with

an adhesive tape.

8. Remove the tip chute and all adapters from the worktable. Remove

the carriage, channeling adapter and riser block from the vacuum

chamber. Clean all parts as described in the QIAcube HT User

Manual.

9. Discard the tip disposal box.

10. Clean any reagents that may have spilled on the instrument

worktable or vacuum chamber with a damp cloth.

11. Discard all waste according to local safety regulations.

Note: for all further cleaning and maintenance operations, see the

QIAcube HT User Manual for detailed instructions.


Optional steps

Vacuum performance check

Using the vacuum performance check option results in one manual

interaction pause after the binding step. This optional setting allows the

user to check whether all the liquid has passed through the

membranes. By default, this step is unchecked.

If the vacuum performance check step is checked, the instrument will

pause after the binding step. The user can then look to see whether

all liquid has passed through the membranes and decide whether to

switch on the vacuum again (Re-do vacuum) or to continue

(Proceed).


1. Open the instrument lid.

Note: The lid sensor is disabled during the vacuum performance

check, allowing to the user to observe the wells.

2. Check the wells on the IndiSpin 96 plate for any remaining liquid.

If no liquid is visible in the wells after the vacuum step, click

Proceed to continue the run.

If liquid remains in the wells, click the Re-do vacuum button to

apply the same vacuum pressure again. The vacuum will be

activated for a certain time or until you press the Stop vacuum

button.

3. Mark any well that is clogged or not empty in the dialog that

appears. This information will be included in the run report. To mark

a well, select the position in the dialog.

To select multiple positions, either press the Shift key and left-click

with the mouse to select adjacent positions, press the CTRL key

and left-click with the mouse to select multiple, nonadjacent

positions, or drag the mouse to select adjacent positions in a

rectangle.

In the position table at the right, check the box next to Marked. The

selected position on the IndiSpin 96 plate will be displayed as

marked

Note: To unmark a position, select the position and uncheck the

box next to Marked.


4. If liquid still remains in any well, manually remove the liquid using a

pipet.

5. After the instrument has added additional reagents, open the hood

to pause the run. Check to see whether the affected well is still

blocked. If so, manually remove the liquid from the affected well

using a pipet.

6. Either click Proceed to continue the run or click Stop run to stop the

run.

Advanced options

Important: INDICAL and QIAGEN do not recommend modifying any of

the parameters found in the Advanced options section.

These parameters have been optimized for each QIAcube HT Kit to

guarantee accurate and valid experiment results. INDICAL and

QIAGEN are not responsible for the outcome and do not support

experiments performed using modified advanced options. Please

note that any changes to these options are carried out at your own

risk.

Note: A warning icon and a corresponding warning message will be

displayed if you change any parameter. The warning text contains the

recommended value. If you return to the recommended value, the

warning message will disappear.


Vacuum parameter

In the Vacuum parameter section, it is possible to change two

parameters: vacuum intensity and vacuum time. The default settings

are 35 kPa for the vacuum intensity and 180 sec for the vacuum time.

The vacuum intensity can be changed from 25 kPa to 70 kPa. Please

note that these changes are not recommended by INDICAL and

QIAGEN. Changing the vacuum intensity parameter only affects the

vacuum intensity following the binding step. All other vacuum steps will

be unaffected.

The vacuum time can be changed from 30 sec to 600 sec. Please note

that these changes are not recommended by INDICAL and QIAGEN.

Changing the vacuum time parameter only affects the vacuum time

following the binding step. All other vacuum steps will be unaffected.

Elution parameter

In the Elution parameter section, it is possible to change the total

elution volume and the elution step. The recommended values for these

parameters are shown in the QIAcube HT Prep Manager Software. The

total elution volume can be changed to another value within the defined

range. Please note that these changes are not recommended by

INDICAL and QIAGEN.

In some cases, it might be helpful to elute multiple times with a lower

volume than one time using the complete elution volume. Increasing

the number of elution steps will result in a multiplication of elution buffer

distribution, incubation pause and vacuum step(s) without influencing

the total amount of elution volume.

The elution parameter can be increased from 1 to 2. Please note that

these changes are not recommended by INDICAL and QIAGEN.


TopElute

TopElute Fluid is used during elution of nucleic acids from the IndiSpin

membrane. It enables application of a stable and high vacuum and

results in equal eluate volumes. In addition,

TopElute Fluid eliminates the formation of drops of elution buffer at the

outlet nozzles of the IndiSpin 96 plates. By default, the Top Elute option

is checked. In case TopElute Fluid should not be used during the run,

uncheck the TopElute option under Advanced options.

Important: Changing the usage of TopElute Fluid is not recommended

or tested by INDICAL and QIAGEN.

Note: TopElute Fluid might be found as a top layer over the elution

buffer. It is inert and has no effects on downstream applications.

Important: Please ensure that you only take the eluate from below the

top layer.


Troubleshooting guide

This troubleshooting guide may be helpful in solving any problems that

may arise.

For more information or help please contact INDICAL Support at

[email protected].

Comments and suggestions

Instrument issues

1 Recovery in case of instrument failure or user interruption

The QIAcube HT interrupts a run upon opening of the hood. The run will proceed normally once the hood is closed. To ensure process safety, this incident is reported in the post-run report.

2 Instrument failure/ cancelled run

It is not possible to recover the run. Please continue manually

3 Blocked membranes When processing samples that might potentially block the membrane, we recommend using a vacuum performance check. If liquid is still visible, remove 500 µl using a pipet. Then scrape the surface of the membrane with a fresh pipet tip in order to relocate any solid particles that may block the membrane. Take care not to damage the membrane. If there is still no liquid flow, pipet all liquid from the well and proceed with the run. After the instrument has added wash buffer, open the hood to pause the run. Check if the well is still blocked. If so, remove all liquid using a pipet. Do not perforate the membrane. Uncovered perforated wells will disturb vacuum integrity during elution across the whole plate. Proceed the run. No buffer will float over from the blocked well into other wells from this step on. Next time, use less sample (tissue).


Little or no pathogen DNA or RNA in the eluate

1 Buffer ACB prepared incorrectly

Check that Buffer ACB concentrate was diluted with the correct volume of Isopropanol, as indicated on the bottle. Use 100% Isopropanol. Repeat the purification protocol with new samples.

2 Buffer AW1 or Buffer AW2 prepared incorrectly

Check that Buffer AW or Buffer AW2 concentrate was diluted with the correct volume of 96–100% ethanol, as indicated on the bottle. Do not use denatured alcohol, which contains other substances such as methanol or methylethylketone. Repeat the purification protocol with new samples.

3 Insufficient sample lysis Proteinase K was stored at elevated temperatures for too long. Repeat the purification procedure using new samples and fresh Proteinase K (see storage recommendations on page 5).

For some DNA viruses and bacteria, heated lysis may improve lysis efficiency. In this case, mix the samples thoroughly after addition of Proteinase K and incubate for 15 min at 70°C.

4 Carrier RNA not added to Buffer VXL mix or degraded Carrier RNA

Please refer to the recommendations for preparation, storage and addition of Carrier RNA.

5 Buffer VXL/Carrier RNA mixture mixed insufficiently

Mix well by pipetting with a large pipette.

6 RNase contamination in Buffer AVE

If tubes containing Buffer AVE are accessed repeatedly, be careful to not introduce RNases which can degrade viral RNA. In case of RNase contamination, replace the open vial of Buffer AVE with a new vial. Repeat the purification procedure with new samples.

7 Nucleic acids in samples already degraded prior to purification

Samples were freeze-thawed more than once or stored at room temperature (15-25ºC) for too long. Always use fresh samples or samples thawed only once. Repeat the purification protocol with new samples.


DNA or RNA does not perform well in downstream applications

1 Little or no DNA or RNA in the eluate

See “Little or no pathogen DNA or RNA in the eluate” (above) for possible reasons.

2 Excessive eluate in the amplification reaction

Some sample types may contain high amounts of background nucleic acids (e.g., animal whole blood, tissue) or PCR inhibiting substances (feces). High amounts of background nucleic acids may inhibit amplification reactions, and removal of inhibitors might not be complete without special treatment. Reduce the amount of sample input or/and the amount of eluate added to the amplification reaction.

3 Too much background nucleic acids in the eluate

Determine the maximum amount of Carrier RNA suitable for your amplification reaction. Adjust the concentration of Carrier RNA solution added to the Buffer VXL accordingly. In RT-PCR, a low DNA background is preferable. Use less eluate

4 Performance of purified nucleic acids in assays varies with aging of reconstituted wash buffers

Salt and ethanol components of Buffer AW1 or Buffer AW2 may have separated out after being left for a ling period between preparation. Always mix buffers thoroughly before each preparation.

Precipitate in buffers

1 Precipitate in Buffer VXL or Buffer ACB

Precipitate may form after storage at low temperature or prolonged storage. To dissolve precipitate, incubate Buffer VXL or ACB for 30 min at 37°C, with occasional shaking.


Order information

Product name Cat. no.

IndiSpin QIAcube HT Kit (480) formerly cadorr Pathogen QIAcube HT Kit

SP54161

QIAcube HT PW (480) formerly QIAcube HT Plasticware

PW950067

intype IC-DNA IC289980

intype IC-RNA IC289970

INDICAL offers a broad range of ready-to-use pathogen specific ELISA

kits, qPCR/ RT-qPCR assays and reagents.

To optimize your workflow, and to handle your sample and throughput

needs, INDICAL additionally offers instruments and kits for the efficient

extraction of nucleic acids from a variety of sample types.

Visit www.indical.com for more information about bactotype, cador,

cattletype, flocktype, IndiMag, IndiSpin, intype, pigtype and virotype

products.

For up-to-date licensing information and product-specific disclaimers,

see the respective INDICAL product handbook or user manual.

Ordering: www.indical.com/contact

Technical Support: [email protected]

Website: www.indical.com

Limited License Agreement for IndiSpin QIAcube HT Pathogen Kit

Use of this product signifies the agreement of any purchaser or user of the product to the following terms:

1. The product may be used solely in accordance with the protocols provided with the product and this handbook and for use with components contained in the kit only. INDICAL grants no license under any of its intellectual property to use or incorporate the enclosed components of this kit with any components not included within this kit except as described in the protocols provided with the product, this handbook, and additional protocols available at www.indical.com. Some of these additional protocols have been provided by INDICAL users for INDICAL users. These protocols have not been thoroughly tested or optimized by INDICAL. INDICAL neither guarantees them nor warrants that they do not infringe the rights of third-parties.

2. Other than expressly stated licenses, INDICAL makes no warranty that this kit and/or its use(s) do not infringe the rights of third-parties.

3. This kit and its components are licensed for one-time use and may not be reused, refurbished, or resold.

4. INDICAL specifically disclaims any other licenses, expressed or implied other than those expressly stated.

5. The purchaser and user of the kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above. INDICAL may enforce the prohibitions of this Limited License Agreement in any Court, and shall recover all its investigative and Court costs, including attorney fees, in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the kit and/or its components.

For updated license terms, see www.indical.com.

Trademarks: bactotype®, cador®, cattletype®, flocktype®, IndiMag®, IndiSpin®, intype®, pigtype®, virotype® (INDICAL BIOSCIENCE GmbH); QIAcube®, QIAGEN® (QIAGEN Group, Hilden, Germany). Registered names, trademarks, etc. used in this document, even when not specifically marked as such, are not to be considered unprotected by law.

HB-2166-EN-002 © 2021 INDICAL BIOSCIENCE GmbH, all rights reserved.

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